Gravitational pull: Real-time reaction monitoring

Ezine

Published: Sep 1, 2014

Author: Steve Down

Channels: Base Peak

Gravitational sampler

The electrospray emitted from the gravitational sampler.
Image: Rapid Communications in Mass Spectrometry

Electrospray ionisation mass spectrometry is generally associated with the detection and identification of compounds, both small and large molecules, but it has also proven useful for studying chemical reactions. When a sampling device is operated continuously and in real time, it can be interfaced with a mass spectrometer to follow the course of a reaction, as signals corresponding to the reactants, intermediates and products are monitored.

Scientists at the National Sun Yat-Sen University in Taiwan have been developing their own sampling device and have released details in Rapid Communications in Mass Spectrometry. Min-Zong Huang, Fu-Jen Hsu, Te-Lin Liu, Amzad Hussain Laskar and Jentaie Shiea explained that their simple version does not need syringe pumps or nebuliser gas because it harnesses the effects of gravity.

They optimised the operational geometry of the device and demonstrated its potential by monitoring two reactions involving small molecules as well as the unfolding of a standard protein.

Simple sampler design relies on gravity

The gravitational sampler consisted of a stainless steel union connector in which one side was used as the reservoir to hold the reaction solutions. A fused silica capillary was attached to the other end of the connector to act as the outlet and sprayer. Using standard solutions of crystal violet, the best electrospray conditions were obtained when the open end of the capillary was 5 mm from the mass spectrometer inlet and 3 mm above it.

In the absence of any applied voltage, a drop of solution formed at the end of the capillary due to the effects of gravity and capillary action. When a voltage of 3 kV was applied to the stainless steel well, a Taylor cone was observed at the capillary outlet and this was transformed into a full-blown electrospray when the voltage was increased to 5 kV. Now, the effects of electroosmotic flow also played a part.

The duration of the electrospray signal increased as the diameter of the capillary was reduced, ranging from 13 to 1.2 minutes for diameters of 25 to 150 µm with 5 µL of solution in the well. The corresponding flow rates varied from 385 to 4167 nL/min, providing a wide range to choose from for studying reaction mechanisms.

However, before going that far, the researchers demonstrated the broad applicability of the sampler by studying separate solutions of the alkaloid reserpine, the peptide angiotensin II and the peptide hormone insulin. Each compound gave the expected electrospray mass spectrum.

Chemical reactions monitored

The first reaction described was a metal chelation. A solution of EDTA was electrosprayed from the source and the signal corresponding to its deprotonated molecule was detected in negative-ion mode. When excess copper sulphate solution was added to the sample well, the deprotonated EDTA signal was accompanied by that of the [EDTA+Cu-3H]– ion, before disappearing completely as the copper ions complexed all of the EDTA. The extracted ion chromatograms of both ions recorded the fall of the EDTA signal and the rise of the chelate, so that the reaction could be followed.

The addition-elimination reaction between 4-aminophenol and acetic anhydride was also monitored. The product could be one of two structural isomers which could not be distinguished from each other since there was no chromatographic separation, a common drawback of direct ionisation methods.

However, a short-lived reaction intermediate was detected and its structure was confirmed by tandem mass spectrometry. Signals for the reactants, intermediate and products were visible in the extracted ion chromatogram.

In the third illustration, unfolding of cytochrome c by the addition of acetic acid was monitored by observing the change in the charge state distribution. Multiply charged ions from +7 to +10 were present for the native protein but they transformed to +13 to +19 for the fully denatured protein as the pH fell. A mixed charge state distribution with ions of +7 to +19 illustrated partial unfolding.

These examples show that the gravitational sampling has the potential to be useful for studying reactions of small and large molecules in real time. Direct delivery of the sample solution from a reservoir to which reactants can be added allows for rapid detection of the products and intermediates. The size and simplicity of the sampler suggest that it will be suitable for use in conjunction with miniature mass spectrometers.